Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by a (9;22) translocation, which generates the constitutively active BCR-ABL1 tyrosine kinase. BCR-ABL1 is the critical mediator of disease pathogenesis, exhibiting constitutive kinase activity that drives survival and proliferation through multiple downstream pathways. While therapy with the ABL1 kinase inhibitor imatinib leads to durable responses in the majority of patients with chronic phase CML, a substantial number of patients develop resistance to therapy, and transient responses are the rule for patients with more advanced disease. Resistance is commonly due to acquired point mutations in the BCR-ABL1 kinase domain, and the second-generation ABL1 inhibitors nilotinib, dasatinib, bosutinib, and ponatinib are largely effective salvage therapies in this therapeutic scenario. However, recent clinical reports suggest that sequential treatment with single agent ABL1 kinase inhibitor therapy can select for BCR-ABL1 compound mutants (two mutations within the same BCR-ABL1 molecule) that confer high-level resistance to multiple inhibitors. In vitro biochemical and cell-based resistance screens, along with crystallographic studies, will be used to better characterize and determine treatment strategies for controlling BCR-ABL1 compound mutations and to minimize the risk of their emergence. Beyond BCR-ABL1 kinase domain mutations, a subset of patients exhibit disease progression via BCR-ABL1 kinase-independent mechanisms, the underlying causative molecular events of which are considerably more heterogeneous and have not been comprehensively characterized. The combination of functional screens using panels of small- molecule inhibitors with whole exome and RNA sequencing analysis will be used to accelerate the identification of molecular mechanisms of BCR-ABL1 kinase-independent resistance. Lastly, despite the utility of ABL1 kinase inhibitor therapies in the management of CML, growing evidence suggests that inhibition of BCR-ABL1 kinase activity in CML stem cells is insufficient to eliminate these cells, thus requiring even the best responding patients to remain on therapy life-long. To develop strategies for selectively targeting CML disease persistence, small-molecule inhibitor-based combinations simultaneously blocking BCR-ABL1 and either bolstering phosphatase activity or inhibiting MEK/ERK-regulated auxiliary signaling will be evaluated in primary CML specimens using a variety of ex vivo cellular, biochemical, and flow cytometric methods. Thus, this proposal centers on: 1) establishment of strategies for controlling and preventing the emergence of highly-resistant BCR-ABL1 compound mutations, 2) elucidation of molecular lesions and therapeutically actionable pathways mediating BCR-ABL1 kinase-independent resistance, and 3) evaluation of mechanisms to selectively eliminate persistent CML stem cells. This knowledge will inform and improve the disease management for patients with CML and other malignancies.